Reporter assays for BER pathway

Abstract

Base excision repair (BER) is one of the most active DNA repair pathways in cells correcting DNA damage from oxidation, deamination, alkylation, and damages induced by free radicals and ionizing radiation. Deregulation or deficiencies in BER mechanisms increase the level of mutations leading to carcinogenesis, and single-strand DNA break formation, which may be converted to double-strand breaks and induce apoptosis. BER deficiency is associated with development of diseases causing neurodegenerative disorders, such as Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). In addition, BER mechanisms can be affected by viral infections, such as HPV, HTLV-1, and HIV-1. Deficiencies in DNA repair in cells can be analyzed using a very convenient and effective approach, where mammalian cells are transfected with plasmids carrying a reporter gene of fluorescent protein that contain inactivating damages. The repair of DNA damages depends on the cellular machinery and is reflected by expression of the reporter gene measured by flow cytometry. In this chapter, we describe this plasmid-based reporter gene system to investigate in cell the repairs of DNA damages involving BER mechanisms.